Microorganisms such as filamentous fungi, yeast and algae produce a variety of lipids, including fatty acyls, glycerolipids, phospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids and prenol lipids. It is advantageous to extract some of these lipids from the microbial cells in which they are produced, and thus a variety of processes have been implemented.
One class of lipids commonly extracted from microbes is glycerolipids, including the fatty acid esters of glycerol (“triacylglycerols” or “TAGs”). TAGs are the primary storage unit for fatty acids, and thus may contain long chain polyunsaturated fatty acids (“PUFAs”), as well as shorter saturated and unsaturated fatty acids and longer chain saturated fatty acids. There has been growing interest in including PUFAs, such as eicosapentaenoic acid [“EPA”; omega-3] and docosahexaenoic acid [“DHA”; omega-3], in pharmaceutical and dietary products. Means to efficiently and cost-effectively extract, refine and purify lipid compositions comprising PUFAs is therefore particularly desirable.
Many typical lipid isolation procedures involve disruption of the microbial cells (e.g., via mechanical, enzymatic or chemical means), followed by oil extraction using organic or green solvents. The disruption process releases the intracellular lipids from the microbial cells, which makes it readily accessible by the solvent during extraction. After extraction, the solvent is typically removed (e.g., by evaporation, for example by application of vacuum, change of temperature or pressure, etc.).
The resulting extracted oil is enriched in lipophilic components that accumulate in the lipid bodies. In general, the major components of the lipid bodies consist of TAGs, ergosterol esters, other sterol esters, free ergosterol and phospholipids. PUFAs present in lipid bodies are mainly as components of TAGs, diacylglycerols, monoacylglycerols, phospholipids and free fatty acids. The extracted oil may then be subsequently refined, to produce a highly purified TAG fraction enriched in PUFAs. Final specifications concerning the purified TAG fraction may be application-dependent, for example, depending on whether the oil is to be used as an additive or supplement (e.g., in food compositions, infant formulas, animal feeds, etc.), in cosmetic or pharmaceutical compositions, etc. Acceptable contaminant standards are either self-imposed (wherein a particular contaminant results in an undesirable property, e.g., haziness/cloudiness, odor) or determined by external nutrition councils (e.g., A Voluntary Monograph Of The Council for Responsible Nutrition [Washington, D.C.], March 2006, specifies the maximum acid, peroxide, anisidine, TOTOX, polychlorinated dibenzo-para-dioxin and polychlorinated dibenzofuran values for omega-3 EPA, omega-3 DHA and mixtures thereof).
U.S. Pat. No. 6,727,373 discloses a microbial PUFA-containing oil with a high triglyceride content and a high oxidative stability. In addition, a method is described for the recovery of such oil from a microbial biomass derived from a pasteurized fermentation broth, wherein the microbial biomass is subjected to extrusion to form granular particles, dried, and the oil is then extracted from the dried granules using an appropriate solvent.
U.S. Pat. No. 6,258,964 discloses a method of extracting liposoluble components contained in microbial cells, wherein the method requires drying microbial cells containing liposoluble components, simultaneously disrupting and molding the dried microbial cells into pellets by use of an extruder, and extracting the contained liposoluble components by use of an organic solvent.
U.S. Pat. Appl. Pub. No. 2009/0227678 discloses a process for obtaining lipid from a composition comprising cells and water, the process comprising contacting the composition with a desiccant, and recovering the lipid from the cells.
U.S. Pat. No. 4,675,132 discloses a process for the concentration of PUFA moieties in a fish oil containing relatively low proportions of saturated and monounsaturated fatty acid moieties of the same chain length as the PUFA moieties to be concentrated, which comprises transesterifying fish oil glycerides with a lower alkanol to form a mixture of lower alkyl fatty acid esters, and extracting said esters with carbon dioxide (CO2) under supercritical conditions.
A process flow diagram developed for a continuous countercurrent supercritical CO2 fractionation process that produces high concentration EPA is disclosed by V. J. Krukonis et al. (Adv. Seafood Biochem., Pap. Am. Chem. Soc. Annu. Meet. (1992), Meeting Date 1987, 169-179). The feedstock for the process is urea-crystallized ethyl esters of menhaden oil, and the basis for the design is a product concentration of 90% EPA (ethyl ester) at a yield of 90%.
Methods in which the distribution of TAGs, diacylglycerols, monoacylglycerols, and free fatty acids can be adjusted in a PUFA-containing lipid composition are sought. Methods for obtaining PUFA-containing lipid compositions which have improved oxidative stability are desired. Methods for obtaining PUFA-containing lipid compositions enriched in TAGs are also desired, as are economical methods for obtaining such compositions.
U.S. Pat. No. 6,166,230 (GIST-Brocades) describes a process for treating a microbial oil comprising PUFAs (e.g., from Mortierella alpina) with a polar solvent to extract at least one sterol (e.g., desmosterol) that is soluble in the solvent and then separating at least some of the solvent containing the sterol from the oil, wherein the oil has a sterol content of less than 1.5%.
U.S. Pat. No. 7,695,626 (Martek) describes a process for recovering neutral lipids comprising PUFAs from a microbial biomass (e.g., Schizochytrium), said process comprising the steps of contacting the biomass with a nonpolar solvent to recover lipid in an extraction process, refining and/or bleaching and/or deodorizing the lipid composition, adding a polar solvent to the lipid composition, cooling the mixture to selectively precipitate at least one other compound (e.g., trisaturated glycerides, phosphorus-containing materials, wax esters, saturated fatty acid containing sterol esters, sterols, squalene, hydrocarbons) and then removing this undesirable compound from the lipid composition.
Previous methods have not utilized techniques of short path distillation as an effective means to avoid exposing PUFAs, specifically highly unsaturated fatty acids, to high temperatures and remove ergosterol (ergosta-5,7,22-trien-3β-ol; CAS Registry Number 57-87-4) contaminants from microbial oils.
Inn Appl. Pub. No. WO 2011/080503 A2 discloses a chromatographic separation process for recovering a PUFA product, from a feed mixture, comprising introducing the feed mixture to a simulated or actual moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as eluent, an aqueous alcohol, wherein the apparatus has a plurality of zones comprising at least a first zone and second zone, each zone having an extract stream and a raffinate stream from which liquid can be collected from said plurality of linked chromatography columns, and wherein (a) a raffinate stream containing the PUFA product together with more polar components is collected from a column in the first zone and introduced to a nonadjacent column in the second zone, and/or (b) an extract stream containing the PUFA product together with less polar components is collected from a column in the second zone and introduced to a nonadjacent column in the first zone, said PUFA product being separated from different components of the feed mixture in each zone. Various fish oil derived feedstocks were purified to produce 85 to greater than 98% EPA ethyl esters. Although Inn Appl. Pub. No. WO 2001/080503 A2 demonstrated processes to recover EPA and DHA in high purity from fish oils, the disclosure also states that suitable feed mixtures for fractionating may be obtained from “synthetic sources including oils obtained from genetically modified plants, animals and microorganisms including yeasts”. Further, “genetically modified yeast is particularly suitable when the desired PUFA product is EPA”.